The overall goal of this program is to better understand how epigenetic mechanisms of genomic regulation contribute to the development and progression of cancer. As an initial effort, we have focused on the development of small molecules tools able to profile and perturb the activity of lysine acetyltransferases (KATs). These enzymes catalyze lysine acetylation, a widespread protein posttranslational modification involved in the regulation of gene expression, DNA repair, protein stability, and metabolism. To better understand the role of protein acetylation in cancer, we have taken a multi-pronged approach. 1. First, we have developed a chemoproteomic method capable of globally profiling cellular KAT activities. Our initial studies lead to the identification of NAT10, an orphan acetyltransferase that is prevalent in cancer cell lines. Currently we are working with collaborators to characterize the biochemical activity of NAT10 and other orphan KAT enzymes, and their relevance in cell growth and proliferation. This chemoproteomic approach has also provided new insights into the sensitivity of lysine acetylation to cellular acetyl-CoA levels, which may have significant implications for diet-induced carcinogenesis and chemoprevention efforts. 2. Second, we have developed a new microfluidic and luminescent oxygen channeling assay sfor the analysis of KAT activity. Currently we are working together with colleagues at the National Center for Advanced Translational Science (NCATS) to adapt these assays to high-throughput format. Our goal is to identify small molecule inhibitors capable of probing the dependency of cancer cells on specific KAT activities. Targeting the cellular acetylation machinery for anticancer treatment is an emerging therapeutic paradigm, and KATs represent a relatively unexplored target relative to HDAC and bromodomains. The development of small molecule probes of specific KAT activities thus has and both basic science and translational implications. 3. Finally, in a collaborative effort we are developing new metabolic tracers for the imaging of cancers exhibiting epigenetic dysregulation due to metabolic mutations. Our goal is to apply simple chemical insights regarding the uptake and solubility of cell-permeating metabolites in order to improve the sensitivity of hyperpolarized 13C imaging agents. By focusing on technologies applicable to the study of these enzyme activities directly in living cells, our studies represent foundational steps towards the identification of novel mechanisms of epigenetic regulation, as well as the development of next-generation therapeutics and diagnostics for cancer treatment.